Factors affecting the binding of antimitotic agents to tubulin have been obtained in detail. Anions, and in particular tartrate, markedly enhance the rate of colchicine binding (a paradoxically slow process) and not dissociation of ligand. This permits accurate estimates of the binding constant which attains a maximum of 3 x 10 to the 7th power M-1. Binding rates for podophyllotoxin and vinblastine are not significantly altered. No conformational changes were detected and it is concluded that effect of the anion is local, near the binding site. Podophyllotoxin binds rapidly (Km 1 x 8 x 10 to the 6th power M-1), is entropy driven, and binds with lower activation energy than colchicine. Podophyllotoxin and colchicine binding are mutually competitive but tropolone interacts only with colchicine binding. Each ligand has two attachment points only one of which is shared by both ligands.